Bone is a specialized dynamic connective tissue that serves the following functions: (a) mechanical, support and site of muscle attachment for locomotion; (b) protective, for vital organs and bone marrow: (c) metabolic; as reserve of ions, especially calcium and phosphate, for the maintenance of serum homeostasis, which is essential for live. To carry out these functions efficiently bone must undergo continuous resorption and renewal, a process collectively known as remodelling. Thus, the mechanical and biological integrity of bone dependents on its continuous destruction (resorption) and continuous rebuilding (formation) at millions of microscopic sites. During adult life bone remodelling is crucial to eliminate and replace structurally damaged or aged bone with structurally new healthy bone. To maintain the proper bone mass resorption and formation are kept in perfect equilibrium. With age the equilibrium between bone resorption and formation becomes altered, often in favor of resorption, resulting in a reduction in bone mass, deterioration of bone architecture, decreased resistance to stress, bone fragility and susceptibility to fractures. The compendium of these symptoms is referred to osteoporosis.
Osteoporosis is a major health problem in Western society. And even though there are other diseases that result in reduction in bone mass, i.e. Paget's disease, osteoporosis is by far the most common and the disease that is the most costly in terms of health care. Since estrogen is a hormone that regulates bone metabolism directly and indirectly, the decrease in estrogen production in post-menopausal women and the decline with age in the production of androgen, which is enzymatically converted to estrogen in men,) is responsible for the risk of osteoporosis, which is estimated to be 85% in women and 15% in men older than 45 years of age. In the United States it is estimated that 17 million post-menopausal women have lost 10% of their peak bone mass, 9.4 million have lost 25% and 5 million have suffered a fracture as a consequence of osteoporosis. Osteoporosis costs America's health care system more than $14 billion a year from spine and hip fractures, which are often the first indication of the disease if it is left undiagnosed.
Osteoporosis, a disease endemic in Western society, typically reflects an imbalance in skeletal turnover, such that bone resorption exceeds bone formation. Bone resorption is a specific function of osteoclasts, which are multinucleated, specialized bone cells formed by the fusion of mononuclear progenitors originating from the hemopoietic compartment, more precisely from the granulocyte-macrophage colony-forming unit (GM-CFU). The osteoclast is the principal cell type, to resorb bone, and together with the bone-forming cells, the osteoblasts, dictate bone mass, bone shape and bone structure. The increased activity and/or numbers of osteoclasts, relative to the activity and or numbers of bone-forming osteoblasts, dictates the development of osteoporosis and other diseases of bone loss.
Even though Paget's disease is not as common or as costly as osteoporosis—it affects 3% of the population over 40, and 10% of the population over 80 years of age—it is nonetheless a significant disease because aside from causing bone fractures it can lead to severe osteoarthritis and severe neurological disorders. Paget's disease is characterized by rapid bone turnover, resulting in the formation of woven bone a tissue type formed initially in the embryo and during growth and which is practically absence from the adult skeleton. Woven bone is marked by brittleness and therefore prone to fractures and bowing. Bones become enlarged and often interfere with blood flow and constrict nerves, resulting in many of the neurological symptoms associated with Paget's disease.
For a disease in which osteoclasts presumably resorb bone at abnormally high levels and osteoblasts form bone at normal levels, as in osteoporosis, the most reasonable therapeutic target would be the osteoclast: decreasing the number of osteoclasts and/or the resorption activity of the osteoclasts, should restore the equilibrium between bone resorption and formation. And, in fact, the treatments now available for osteoporosis are intended to suppress bone resorption.
Osteoclasts are derived from the monocyte-macrophage family. Upon stimulation of the CFU-GM with macrophage colony stimulating factor (M-CSF) form promonocytes which are immature nonadherent progenitors of mononuclear phagocytes and osteoclasts. The promonocytes, may proliferate and differentiate along the macrophage pathway, eventually forming a tissue macrophage, or may differentiate along the osteoclast pathway, depending on the cytokines to which they become exposed. For example, the receptor activator NF-κB ligand (RANKL) (Simonet W S, Lacey D L, Dunstan .R, Kelley M, Chang M-S, Luethi R et al 1997 Osteoprotegerin, a novel secreted protein involved in the regulation of bone density. Cell 89:309–319) a cytokine expressed on the membrane surface of osteoblasts influences promonocytes to differentiate into osteoclasts rather than macrophages, while treatment with M-CSF drives the promonocyte to develop into macrophages. Since M-CSF and other cytokines i.e., interleukin-1 or TNF-α, that support expression of RANKL are products of macrophages it may be assumed that immunomodulating substances, which alter the expression of, these cytokines and growth factors, may affect not only macrophages but also osteoclasts.
It has long been known that beta glucans, and particularly the beta glucans from yeast, activate macrophages and have profound effects on the synthesis and levels of many cytokines, which in turn are responsible for modulating the function of many other cells. (Stoy, Y. “Macrophage biology and pathobiology in the evolution of immune responses: a functional analysis,” Pathobiology, 69:179–211, 2001; Underhill D M, Ozinshy, A. “Phagocytosis of microbes: complexity in action,” Annu Rev Immunol. 20:825–52, 2002; Purohit A, Newman S P, Reed M J. “The role of cytokines in regulating estrogen synthesis: implications for the etiology of breast cancer,” Breast Cancer Res 4:65–69, 2002; Ismail N, Olano J P, Feng H M, Walker D H. “Current status of immune mechanisms of killing intracellular organisms” FEMS Microbiol Lett 207:111–120, 2002; Hubel K, Dale D C, Liles W C. “Therapeutic use of cytokines to modulate phagocyte function for the treatment of infectious diseases: current status of granulocyte colony stimulating factor, granulocyte-macrophage stimulating factor, macrophage colony stimulating factor and interferon gamma” J. Inf Dis 185:1490–1501, 2002.).
Even though there are a number of therapeutic modalities for osteoporosis, which include bisphosphonates (Fleisch H, “Development of biphosphonates,” Breast Cancer Res. 4:30–34, 2002), estrogen (Spencer, C P, Stevenson. J C “Oestrogen and anti-oestrogen for the prevention and treatment of osteoporosis.” In Osteoporosis: Diagnosis and Management, Martin Muniz, England, 1998, pp 111–123), or “Selective Estrogen Receptor Modulators,” (SERMS) most of these have significant undesirable side-effects.
Glucans are polysaccharides consisting of glucose subunits. β-(1,6) branched β-(1,3) glucan is a naturally occurring class of polysaccharides that can be extracted from Baker's yeast and other yeast species, mushrooms, plants and some bacterial, lichen and algal species (reviewed in Chemistry and Biology of (1→3)-β-Glucans, B. A. Stone and A. E. Clarke, 1992, La Trobe University Press, Australia). β-(1, 6) branched (1,3) glucans have been shown to have immune enhancing and cholesterol-lowering capabilities. Yeast synthesizes at least three different types of beta glucans, a linear β-1,3-D-glucans, a linear β-1,6-D-glucan and a β3-(1,6) branched β-1,3-(1,3) glucan. However, linear β-1,3-1,3-D and linear β-1,6-D-glucans do not activate or only marginally activate macrophages, NK cells or neutrophils.
As a class of polysaccharides, β-(1,6) branched β-(1,3) glucans are composed of a main chain of glucose subunits linked together in and branches linked to the main chain by a (1→6) β glycosidic linkage. Yeast β-(1,6) branched β-(1,3) glucan is composed of mostly of a main chain of glucose units linked by (1→3) beta glycosidic linkages (90% or more) with a variable number of relatively short side chains linked by β-(1→6) glycosidic linkages (10% or less); the chemical name for this glucan is poly-(1,3)-β-D-glucopyranosyl-(1,6)-β-D-glucopyranose. There are several different types of beta glucans, which vary in backbone composition, branching, type of monomers or substituents, resulting in polysaccharides that have very different physical and biological properties (Metz, Ebert, and Weicher, Chromatographia 4:345, 1970; Manners et al., The structure of β-(1-3) D-glucan from yeast cell walls. Biochem. J. 135:19, 1973; U.S. Pat. No. 5,223,491).
Whereas all the β-1,3/1,6-D-glucans have been shown to activate the immune system of vertebrate as well as invertebrate organisms, the yeast-derived β-1,3/1,6-D-glucan is a most powerful activator of macrophages, NK cells, and neutrophils. Beta glucan from yeast activates the immune system by binding to a specific receptor on the cell membrane of macrophages (Czop and Kay, Isolation and characterization of β-glucan receptors on human mononuclear phagocytes. J. Exp. Med. 173:1511–1520, 1991). The activated macrophages increase their phagocytic and bactericidal activities as well as the production of a wide range of cytokines (Burgaletta, C and Golde, D W, in Immune Modulation and control of neoplasia by adjuvant Therapy (Chirigos, M. A., ed), pp 195–200. Raven Press, NY, 1978; Sherwood et al., “Glucan stimulates production of antitumor cytolytic/cytostatic factors by macrophages,” J. Biol Resp. Mod., 6:358–381; Sherwood, et al., “Enhancement of interleukins 1, and interleukins 2 production by soluble glucan”; Browder et al., “Beneficial effects of enhanced macrophage function in the trauma patient,” Ann. Surg. 211:605–613). Enhanced function of macrophages, as well NK cells, appear responsible for a number of beneficial effects of yeast beta glucan, such as increased resistance of the host to infection by bacteria, viruses, fungi and protozoan parasites (Williams et al., “Protective effect of glucan in experimentally induced candidiasis,” J Reticuloendot. Soc. 23:479–490, 1978; Williams and DiLuzio. “Immunopharmacological modification of experimental viral diseases by glucan,” EOS JK Immunol Immunopharmacol 5:78–82, 1985; Babineau et al. “A phase II multicenter, double blind, randomized, placebo-controlled study of three dosages of an immunomodulator (PCG-glucan) in high risk surgical patients,” Arch. Surg., 129:601–609., 1994). In addition, the enhanced function of macrophages and NK cells appears to increase the host defenses against malignant tumors (Mansell et al. “Macrophage mediated destruction of human malignant cells in vivo,” J Natl Canc. Inst. 54:571–576, 1975; Williams et al. “Chemoimmunotherapy of experimental hepatic metastasis,” Hepatology, 7:1296–1304, 1985; Ueno. “Beta-1,3-D-glucan, its immune effect and its clinical use,” Jap. J Soc. Terminal Systemic Dis. 6:151–154, 2000).
Beta-1,3/1,6-D-glucan isolated from baker's or brewer's yeast (Saccharomyces cerevisiae strain) as well other yeasts, is insoluble, and furthermore the variability in the number of beta-(1,6) side chains makes it extremely difficult if not impossible to determine whether the beta-1,3/1,6-D-glucan is the branched beta-1,3/1,6-D-glucan or a mixture of beta-1,3-D-glucan plus beta-1,6-D-glucan, or a mixture of all three beta glucans. Yeast makes all three types of beta glucans. Since only the branched beta-1,3/1,6-D-glucan activates macrophages, it would be desirable to have pure beta-1,3/1,6-D-glucan; in addition, insoluble beta-1,3/1,6-D-glucan is difficult to formulate for parenteral or topical administration. It would be desirable to have a beta glucan that could be easily characterized, and which could be easily formulated for topical and parenteral administration. In addition, it would be of benefit for formulation purposes to have a lower molecular weight beta glucan that retains biological activity. A low molecular weight, soluble beta-1,3/1,6-D-glucan used topically would also be able to penetrate faster and, used parenterally, would very likely reach tissue macrophages faster, resulting in an earlier activation.
To date the soluble beta glucans that have been available are all of the high molecular weight variety, and for the major part these glucans were made soluble by chemical modifications or solubilized by sequential treatments with alkali/acid/alkali. A number of soluble glucans have been obtained by derivatization of the natural, insoluble beta-1,3/1,6-D-glucan compound, such as phosphorylation (U.S. Pat Nos. 4,739,046; 4,761,402), sulfation, amination (U.S. Pat. No. 4,707,471) or methylation. A beta-1,3/1,6-D-glucan solubilized by sequential treatment with alkali/acid/alkali of insoluble beta-1,3/1,6-D-glucan (U.S. Pat. No. 5,849,720) has been shown to be effective in humans to control infections in surgical patients (Babineau et al. A phase II multicenter, double blind, randomized, placebo-controlled study of three dosages of an immunomodulator (PCG-glucan) in high-risk surgical patients (Arch. Surg., 129:601–609, 1994).
There is therefore a need for therapies to inhibit or prevent bone loss that have less or no side effects and offer more natural biological mechanisms.